WO2016158479A1 - Congélateur et procédé de congélation - Google Patents

Congélateur et procédé de congélation Download PDF

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Publication number
WO2016158479A1
WO2016158479A1 PCT/JP2016/058634 JP2016058634W WO2016158479A1 WO 2016158479 A1 WO2016158479 A1 WO 2016158479A1 JP 2016058634 W JP2016058634 W JP 2016058634W WO 2016158479 A1 WO2016158479 A1 WO 2016158479A1
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Prior art keywords
spray
freezing
fine solid
cryogenic
cryopreservation container
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PCT/JP2016/058634
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English (en)
Japanese (ja)
Inventor
淳 石本
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国立大学法人東北大学
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Publication of WO2016158479A1 publication Critical patent/WO2016158479A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/36—Freezing; Subsequent thawing; Cooling
    • A23L3/37—Freezing; Subsequent thawing; Cooling with addition of or treatment with chemicals
    • A23L3/375—Freezing; Subsequent thawing; Cooling with addition of or treatment with chemicals with direct contact between the food and the chemical, e.g. liquid nitrogen, at cryogenic temperature
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/30—Fish eggs, e.g. caviar; Fish-egg substitutes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a freezing apparatus and a freezing method.
  • Stem cells which have a self-replicating function and a property of differentiating into other cells, are expected to make great industrial contributions, for example, because they serve as a source of human cells.
  • Known methods for cryopreserving cells such as stem cells include slow freezing and vitrification freezing.
  • a general slow freezing method cells are suspended in a storage solution to which a cryoprotectant is added, and the temperature is slowly lowered and stored at a liquid nitrogen temperature.
  • the cooling rate it is necessary to appropriately control the cooling rate. If the cooling rate is not appropriate, a large amount of ice crystals may be formed on the cells at a certain timing, and the cells may be physically damaged.
  • the slow freezing method is not suitable for cryopreservation of primate embryonic stem cells (ES cells: Embryonic Stem Cells), iPS cells (induced pluripotent stem cells), germ cells, etc., and cell viability upon thawing Is 1% or less, indicating a very low value. Vitrification is used for these cells (see, for example, Patent Document 1 and Patent Document 2).
  • vitrification freezing method cells are suspended in a vitrification preservation solution containing a special cryoprotective solution with a high concentration, and the cells are accommodated in a container, and within about 15 seconds from the start of cell suspension.
  • This is a method of immersing in liquid nitrogen, rapidly cooling to below the glass transition point, and solidifying and freezing in an amorphous glass state without crystallizing intracellular and external moisture.
  • this vitrification freezing method there is no volume expansion of water and there is little damage to cells. It should be noted that when thawing the vitrified cells, it is necessary to add a warm medium and rapidly thaw to avoid recrystallization of water.
  • vitrification freezing method cells are suspended in a cryoprotective solution and frozen by spraying liquid nitrogen into a container containing the cells, a method in which a container containing the suspension is directly poured into liquid nitrogen and frozen, Etc. are known (see, for example, Patent Document 2).
  • the conventional general vitrification freezing method requires a cryopreservation solution having a high solute concentration, and thus has a problem that the cytotoxicity due to the solute is high.
  • the cooling rate is about ⁇ 72 ° C./min, and a container containing cells is directly put into liquid nitrogen.
  • the cooling rate is about ⁇ 300 ° C./min, and the temperature drop rate is relatively small.
  • the present invention has been made in view of the above-described problems, and provides a freezing apparatus that vitrifies and freezes cells rapidly with a spray flow containing cryogenic fine solid particles at the time of freezing so that a high cell viability can be obtained upon thawing.
  • a freezing device for vitrifying cells with a small amount of cryopreservation solution to provide a freezing device with a high temperature drop rate, to provide a freezing method for the freezing device, etc. .
  • the freezing apparatus of the present invention has at least the following configuration.
  • a freezing device for elastic membrane capsules containing moisture as a freezing object A cryopreservation container containing the object to be frozen;
  • the freezing method of the present invention comprises at least the following configuration.
  • a freezing method of a freezing apparatus for elastic membrane capsules containing water as a freezing object The freezing device includes a cryopreservation container that houses the object to be frozen, Spraying a spray stream containing cryogenic fine solid particles in the cryopreservation container, Support the cryopreservation container containing the object to be frozen on the downstream side of the spray flow by the support, The spray unit continuously sprays a spray flow containing cryogenic fine solid particles on the cryopreservation container to vitrify and freeze the object to be frozen stored in the cryopreservation container.
  • the freezing apparatus which vitrifies and freezes freezing objects, such as a cell, rapidly can be provided by the spray flow containing a cryogenic fine solid particle at the time of freezing so that it may become a high cell viability at the time of thawing
  • the freezing apparatus with a large temperature fall rate can be provided.
  • a method for freezing the freezing apparatus can be provided.
  • the front view which shows an example of the freezing apparatus which concerns on embodiment of this invention.
  • generation apparatus which provided the spiral nozzle at the front-end
  • generation apparatus (a) is a figure which shows an example when not applying an ultrasonic wave to a Laval nozzle part,
  • FIG. 4B is a diagram illustrating an example when ultrasonic waves from an ultrasonic vibrator are applied to a Laval nozzle portion.
  • generation apparatus (a) is an example when not applying the ultrasonic wave by an ultrasonic transducer to a Laval nozzle part
  • the conceptual diagram which shows an example of the freezing apparatus which concerns on embodiment of this invention.
  • a freezing apparatus includes a cryopreservation container that contains a subject to be frozen, such as cells, and a cryopreservation container that continuously sprays a spray flow containing cryogenic fine solid particles at a high speed to form a cryopreservation container. It has a cryogenic fine solid particle continuous generation device as a spraying section for vitrifying and freezing objects to be frozen such as contained cells.
  • the object to be frozen is not particularly limited as long as it can be vitrified and frozen by the freezing apparatus according to the embodiment of the present invention.
  • the elastic membrane sac containing water such as cells, specifically iPS cells, ES Examples thereof include cells, germ cells, living tissues, blood, plant cells, and foods (frozen foods).
  • the cryogenic fine solid particle continuous generator may be a one-component cryogenic fine solid particle continuous generator or a two-component cryogenic fine solid particle continuous generator.
  • the one-component cryogenic fine solid particle continuous production apparatus continuously sprays a spray stream containing, for example, a cryogenic fine solid nitrogen particle at a high speed.
  • generation apparatus may spray the spray flow of cryogenic fine solid particles, such as a carbon dioxide, argon, and hydrogen, instead of nitrogen.
  • a two-component cryogenic fine solid particle continuous production device is a spray flow containing cryogenic fine solid nitrogen particles produced by mixing two fluids such as cryogenic helium gas as a cryogen and supercooled liquid nitrogen in a nozzle. Spray continuously at high speed.
  • the cryogenic fine solid particles produced by the two-component cryogenic fine solid particle continuous production apparatus may be composed of any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more. Good.
  • the embodiment of the present invention includes the contents shown in the drawings, but is not limited to this.
  • generation apparatus is employ
  • FIG. 1 is a front view showing an example of a cryogenic fine solid particle continuous generation device 100 (fine particle generation device) as a cryopreservation container 60 and a spray section of a freezing device 200 according to an embodiment of the present invention.
  • FIG. 2 is a diagram illustrating an example of a main part of the fine particle generation apparatus 100.
  • FIG. 3 is an enlarged view of a main part of the fine particle generating apparatus 100 shown in FIG.
  • FIG. 4 is an enlarged cross-sectional view of the vicinity of the nozzle of the fine particle generating apparatus 100.
  • the cryopreservation container 60 accommodates objects to be frozen such as cells 61.
  • the support device 70 supports the cryopreservation container 60 on the downstream side of the spray flow sprayed by the cryogenic fine solid particle continuous production device 100.
  • the fine particle generator 100 continuously generates one-component cryogenic fine solid particles by using a cryogenic supercooled liquid and a cryogenic gas composed of the same element as the supercooled liquid.
  • the fine particle generating apparatus 100 is provided downstream of the mixing unit 10, the mixing unit 10 that generates a one-component mixed phase flow by mixing a supercooled liquid and a high-speed flow of a cryogenic gas, and the mixing thereof.
  • a Laval nozzle unit 11 that is a nozzle 1 that generates a spray flow including cryogenic fine solid particles from the one-component mixed phase flow generated in the unit 10.
  • cryogenic gas nitrogen is employed as the cryogenic gas composed of the same elements as the supercooled liquid nitrogen.
  • a two-phase flow (LN 2 -GN 2 ) of supercooled nitrogen liquid (LN 2 ) and cryogenic nitrogen gas (GN 2 ) is discharged from the reduced diameter portion 11b (throat portion) to the injection portion 11c (expanded portion). passes through the opening portion), by solid phase formation based on adiabatic expansion is performed, is generated fine solid nitrogen particles (SN 2), the spray flow is injected containing fine solid nitrogen particles (SN 2) .
  • the frame 210 is provided with the fine particle generating device 100 in the vicinity of the upper portion thereof, and the support device 70 for supporting the cryopreservation container 60 that stores the cells 61 and the like is provided in the substantially central portion. It has been.
  • the cryopreservation container 60 is supported by the support device 70 so as to be movable in a predetermined direction and / or to be rotatable.
  • the nozzle 1 for ejecting a spray flow containing cryogenic fine solid particles is disposed below the fine particle generating apparatus 100.
  • the nozzle 1 is configured to inject a spray flow toward the cryopreservation container 60.
  • the nozzle 1 is provided with a liquid nitrogen conduit 3, a nitrogen gas conduit 4, and the like.
  • the liquid nitrogen conduit 3 supplies liquid nozzle (LN 2 ) supercooled to a cryogenic temperature to the nozzle 1 from, for example, a liquid nitrogen tank (not shown).
  • the liquid nitrogen conduit 3 includes a valve 3a, and the supply amount and pressure of the liquid nitrogen can be controlled by the valve 3a.
  • the nitrogen gas conduit 4 supplies cryogenic nitrogen gas (GN 2 ) to the nozzle 1 from a nitrogen gas tank (not shown), for example.
  • the nitrogen gas conduit 4 includes a valve 4a, and the supply amount of nitrogen gas (GN 2 ) can be controlled by the valve 4a.
  • the nozzle 1 is configured such that all or part of the nozzle 1 is accommodated in the heat insulating portion 5, and the tip portion of the nozzle 1 protrudes outside the heat insulating portion 5.
  • the heat insulation part 5 has a vacuum heat insulation structure so as to insulate the nozzle 1 from the outside air.
  • the vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. Yes.
  • the movable dish portion 7 is provided below the nozzle 1.
  • the movable dish part 7 is disposed between the nozzle 1 and the cryopreservation container 60, the spray flow from the nozzle 1 to the cryopreservation container 60 and the radiation of cold air are prevented, and the movable dish part 7 is disposed at other positions.
  • the movable dish portion 7 is configured so that the spray flow from the nozzle 1 is sprayed onto the cryopreservation container 60.
  • the cryogenic fine solid particle continuous generation apparatus 100 employs a concentric mixing type high-speed two-fluid nozzle.
  • the nozzle 1 includes a mixing unit 10, a laval nozzle unit 11, and the like.
  • the mixing unit 10 of the nozzle 1 has an inner tube 14 and an outer tube 15 that are concentrically combined.
  • the outer diameter of the inner tube 14 is configured to be smaller than the inner diameter of the outer tube 15.
  • a gap 45 is formed between the inner tube 14 and the outer tube 15.
  • the inner tube 14 communicates with the nitrogen gas conduit 4, and the distal end portion 14 a of the inner tube 14 is formed in a tapered shape.
  • a communication portion 15 a communicating with the liquid nitrogen conduit 3 is formed on the side wall of the outer tube 15, and the communication portion 15 a is connected to an opening portion 15 b formed on the inner wall of the outer tube 15.
  • the opening 15b is configured to be located near the side surface in the vicinity of the distal end portion 14a of the inner tube 14.
  • cryogenic nitrogen gas (GN 2 ) is jetted at high speed from the distal end portion 14 a of the inner tube 14.
  • the supercooled liquid (LN 2 ) is supplied to the gap 45 between the inner tube 14 and the outer tube 15 through the liquid nitrogen conduit 3, the communication portion 15a, and the opening portion 15b.
  • the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of the cryogenic gas (GN 2 ) are mixed in the mixing section 10 near the downstream side of the distal end portion 14 a of the inner tube 14, and a one-component mixed phase is obtained.
  • a stream (LN 2 -GN 2 ) is generated.
  • This one-component mixed phase flow (LN 2 -GN 2 ) is introduced into a Laval nozzle unit 11 provided downstream of the mixing unit 10.
  • the Laval nozzle portion 11 is provided on the downstream side of the mixing portion 10 and in the vicinity of the distal end portion of the outer tube 15.
  • the Laval nozzle part 11 has the introduction part 11a, the diameter reducing part 11b (throat part), and the injection part 11c (expansion part).
  • the introduction part 11 a is formed so that the inner diameter on the upstream side is substantially the same as the inner diameter of the outer tube 15 of the mixing part 10.
  • a high-speed one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a.
  • the reduced diameter portion 11b is provided on the downstream side of the introduction portion 11a, and is formed to have an opening cross-sectional area smaller than the opening cross-sectional area of the introduction portion 11a. Specifically, the reduced diameter portion 11b is formed in a shape in which the inner diameter decreases as it approaches the minimum inner diameter portion of the reduced diameter portion 11b from the introduction portion 11a.
  • the injection part 11c (expanded part) is provided on the downstream side of the reduced diameter part 11b, and is formed to have an opening sectional area larger than the opening sectional area of the reduced diameter part 11b. Specifically, the injection portion 11c is formed in an expanded shape in which the opening cross-sectional area increases from the reduced diameter portion 11b toward the downstream side.
  • the high-pressure / high-speed one-component mixed phase flow has a velocity equal to or lower than the sound velocity, and the velocity increases as the inner diameter decreases from upstream to downstream.
  • the one-component mixed phase flow is substantially sonic.
  • the opening cross-sectional area increases from the reduced diameter part 11b to the downstream end part of the injection part 11c, the adiabatic expansion of the one-component multiphase flow causes the flow to exceed the speed of sound, and the ice nucleus grows.
  • One-component cryogenic fine solid particles are generated, and a spray flow containing the one-component cryogenic fine solid particles is continuously ejected from the ejection unit 11c. Further, the high-pressure / high-speed one-component mixed phase flow is extremely low temperature, and the one-component mixed phase flow adiabatically expands from the reduced diameter portion 11b of the Laval nozzle portion 11 to the downstream end portion of the injection portion 11c. Becomes a speed exceeding the speed of sound, and the temperature is significantly lowered as compared with the introduction part 11a, and the generation of one-component cryogenic fine solid particles is promoted.
  • the inner diameter of the outer tube 15 of the mixing unit 10 described above is about 2.5 mm
  • the outer diameter of the inner tube 14 is about 1.4 mm
  • the inner diameter of the inner tube 14 is about 0.5 mm
  • the introduction portion 11a of the Laval nozzle unit 11 The inner diameter of the upstream end portion is about 2.5 mm
  • the inner diameter of the reduced diameter portion 11b is about 1.0 mm
  • the inner diameter of the tip portion of the injection port of the injection portion 11c is about 2.2 mm.
  • Each size of the mixing part 10 and the Laval nozzle part 11 is not restricted to the said form, It is preferable to set suitably.
  • the cryogenic fine solid particle continuous generation apparatus 100 includes the ultrasonic transducer 6.
  • the ultrasonic transducer 6 applies ultrasonic waves to the Laval nozzle unit 11 as shown in FIGS.
  • the ultrasonic wave generated by the ultrasonic vibrator 6 applies ultrasonic waves to the Laval nozzle unit 11 as shown in FIGS.
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle part 11, and ice of one-component cryogenic fine solid particles (SN 2 particles) is generated.
  • Nucleation can be promoted, and refinement of a substantially spherical one-component cryogenic fine solid particle (SN 2 particle) having a fine uniform particle diameter can be promoted.
  • Cavitation is a physical phenomenon in which bubbles are generated and disappeared in a short time due to a pressure difference in a fluid.
  • a short-lived high-temperature, high-pressure local field hot spot
  • the refined SN 2 particles are formed in a fine substantially spherical shape.
  • the ultrasonic transducer 6 includes an ultrasonic vibration generation unit 6a and an ultrasonic transmission unit 6b.
  • the ultrasonic vibration generation unit 6a generates ultrasonic waves having a specified frequency and a specified amplitude under the control of a control device (not shown).
  • the ultrasonic transmission unit 6 b is configured by a substantially rod-shaped metal member, and transmits the ultrasonic wave generated by the ultrasonic vibration generation unit 6 a to the Laval nozzle unit 11.
  • the ultrasonic wave applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 has, for example, a frequency of about 30 kHz to 2 MHz and an amplitude of about 10 ⁇ m to 50 ⁇ m, preferably a frequency of about 40 kHz to 950 kHz and an amplitude. It is about 20 ⁇ m to 40 ⁇ m, and optimally, 45 kHz and an amplitude of about 30 ⁇ m.
  • the frequency and amplitude of the ultrasonic wave applied to the Laval nozzle unit 11 are appropriately set according to the particle size, number, etc. of the one-component cryogenic fine solid particles generated by the Laval nozzle unit 11.
  • ultrasonic waves applied from the ultrasonic transducer 6 to the Laval nozzle unit 11 are ultrasonic waves having a high frequency (megasonic) of about 1 MHz to several tens of MHz, or several tens of MHz to several hundreds of MHz, for example, extremely low temperature
  • a high frequency megasonic
  • the ice nucleation and atomization promoting characteristics of fine solid particles are further improved.
  • the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied is preferably near the position where ice nucleation of one-component cryogenic fine solid particles of the Laval nozzle unit 11 is performed. A position slightly downstream from the diameter portion 11b is preferable. Further, the position where the ultrasonic wave generated by the ultrasonic transducer 6 is applied may be an arbitrary position of the injection portion 11c on the downstream side from the reduced diameter portion 11b of the Laval nozzle portion 11, or in the entire Laval nozzle portion 11. It may also be set as appropriate according to the particle diameter, the number, etc. of the one-component cryogenic fine solid particles produced by the Laval nozzle unit 11.
  • the ultrasonic transducer 6 may apply ultrasonic waves directly or indirectly to the Laval nozzle unit 11 or may apply the ultrasonic wave to the Laval nozzle unit 11 from within the heat insulating unit 5 as a vacuum heat insulating unit.
  • Cryogenic supercooled liquid nitrogen (LN 2 ) is introduced from a liquid nitrogen tank (not shown) into the mixing unit 10 in the outer tube 15 through the liquid nitrogen conduit 3, the communicating portion 15a of the outer tube 15, and the opening 15b.
  • the High-pressure and high-speed cryogenic nitrogen gas (LN 2 ) is introduced from a nitrogen gas tank (not shown) into the mixing unit 10 through the nitrogen gas conduit 4 and the inner tube 14.
  • the pressure of the cryogenic nitrogen gas (LN 2 ) is, for example, about 0.1 MPa to 1.0 MPa, and in this embodiment is about 0.4 MPa.
  • the pressure of the cryogenic nitrogen gas (LN 2 ) may be about 0.5 MPa to 1000 MPa or about 1.0 to 10 MPa.
  • high-pressure and high-speed cryogenic nitrogen gas (GN 2 ) is jetted at a high speed from the distal end portion 14 a of the inner tube 14, and supercooled liquid (LN 2 ) is discharged from the inner tube 14. It is introduced into the mixing unit 10 through the gap 45 between the outer tubes 15, and in the mixing unit 10, the supercooled liquid nitrogen (LN 2 ) and the high-speed flow of cryogenic gas (GN 2 ) are mixed to form one component. A multiphase flow (LN 2 -GN 2 ) is generated.
  • the high-pressure, high-speed, low-temperature, one-component mixed phase flow (LN 2 -GN 2 ) generated in the mixing unit 10 is introduced into the introduction unit 11a of the Laval nozzle unit 11, and at the minimum inner diameter part of the reduced diameter unit 11b.
  • the one-component multiphase flow becomes substantially sonic velocity, and the flow crosses the sonic velocity due to adiabatic expansion of the one-component multiphase flow as the opening cross-sectional area increases from the reduced diameter portion 11b to the downstream end portion of the injection portion 11c.
  • the one-component multiphase flow adiabatically expands in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles (SN 2 particles) is continuously generated.
  • ultrasonic waves about 45 kHz, amplitude of about 30 ⁇ m
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11
  • one-component cryogenic temperature is generated.
  • Formation of ice nuclei of fine solid particles (SN 2 particles) can be promoted, and miniaturization of one-component cryogenic fine solid particles (SN 2 particles) having a substantially uniform spherical particle shape can be promoted.
  • FIG. 5 shows a cooling heat flow of cryogenic fine solid nitrogen particles (SN 2 ) generated by cryogenic nitrogen gas (GN 2 ) and supercooled liquid nitrogen (LN 2 ) by the cryogenic fine solid particle continuous production apparatus 100.
  • SN 2 cryogenic fine solid nitrogen particles
  • LN 2 supercooled liquid nitrogen
  • the cooling heat flow flux value q w is rapidly increases in a short time reach the maximum cooling heat flux value, then decreased gradually.
  • the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus has a heat flux of 10 5 W / m 2 level and has a very high cooling ability.
  • FIG. 6 is a diagram showing an example of a spray collision pressure value of a one-component cryogenic fine solid nitrogen particle (SN 2 ) and a spray collision pressure value by simulation.
  • the horizontal axis (x axis) represents the dimensionless time t *
  • the vertical axis (y axis) represents the pressure p * obtained by making the collision pressure dimensionless.
  • one-component cryogenic fine solid nitrogen particles (SN 2 particles) are caused to collide with a piezoelectric piezoelectric pressure sensor, and the spray collision pressure value of the SN 2 particles is indicated by a dotted line.
  • the pressure of the nitrogen gas (GN 2 ) tank was set to 0.4 MPa, and the pressure of the liquid nitrogen (LN 2 ) tank was set to 0.2 MPa.
  • the collision pressure value obtained by single particle collision numerical calculation (CFD) is shown by a solid line.
  • the collision pressure p * increased rapidly and then decreased.
  • the numerical calculation result shows that the collision pressure p * tends to rise and decrease immediately after the collision.
  • FIG. 7 is a diagram illustrating an example of the nozzle 1 of the cryogenic fine solid particle continuous generation apparatus 100 including the spiral nozzle 18 at the tip of the Laval nozzle.
  • a spiral nozzle 18 as shown in Japanese Patent Application Laid-Open No. 2011-171691 is provided at the tip of the Laval nozzle portion 11 of the nozzle 1.
  • SN 2 particles one-component cryogenic fine solid nitrogen particles
  • PIA Physical Imaging Techniques.
  • PIA for example, as shown in FIG. 7, in a defined region CR (Control region), SN 2 particles flying are magnified using a microscope lens and the particle size is analyzed by image analysis. And measure the speed.
  • a two-color laser for example, a dual pulse YAG laser, a dye laser for depth of field check, a high-resolution color camera for PIA, PIA image analysis software, or the like
  • a high-load distributed computing (Grid Computing) method based on massively parallel computation using a cluster-type high-speed workstation, and the atomization characteristics of the nozzle, for example, the particle size distribution
  • the number density distribution, flow velocity / temperature distribution, etc. can be appropriately and quantitatively evaluated.
  • a PTV (Particle Tracking Velocimetry) algorithm can be used for quantifying the particle velocity.
  • the PTV algorithm to be used considers a distribution pattern of a particle image group composed of a target particle image and a particle image in the vicinity thereof, and performs particle tracking using the similarity.
  • FIG. 8 is a diagram showing an example of the particle size distribution of one-component cryogenic fine solid particles produced by the cryogenic fine solid particle continuous production apparatus 100.
  • FIG. 8A is a diagram illustrating an example in which ultrasonic waves are not applied to the Laval nozzle unit 11
  • FIG. 8B is an example in which ultrasonic waves from an ultrasonic transducer are applied to the Laval nozzle unit 11.
  • FIG. 7 the analysis by the PIA-PTV was performed in a specified region (horizontal 0.92 mm, vertical 0.7 mm) as a visual field from a position below 4.5 mm from just below the nozzle hole.
  • the horizontal axis (x axis) represents the particle diameter d p [ ⁇ m]
  • the left vertical axis (y 1 axis) represents the frequency f f [%]
  • the right the vertical axis (y 2 axis) shows the cumulative f a [%].
  • the average particle size was reduced by about 2.5% in the case where the ultrasonic transducer was installed in the nozzle (ULA) compared to the case where no ultrasonic wave was applied (non-ULA). Specifically, when the ultrasonic wave by the ultrasonic vibrator is not applied to the Laval nozzle part (non-ULA), the average particle diameter is 4.1 ⁇ m, and when the ultrasonic wave by the ultrasonic vibrator is applied to the Laval nozzle part (ULA) ), And the average particle size was 3.9 ⁇ m.
  • FIG. 9 is a diagram illustrating an example of a particle velocity distribution of one-component cryogenic fine solid particles generated by the cryogenic fine solid particle continuous production apparatus 100.
  • FIG. 9A is a diagram showing an example when ultrasonic waves from an ultrasonic transducer are not applied to the Laval nozzle portion 11
  • FIG. 9B is a diagram showing an example when ultrasonic waves are applied to the Laval nozzle portion.
  • the horizontal axis (x axis) indicates the particle velocity V p [m / s]
  • the left vertical axis (y 1 axis) indicates the frequency f f [%].
  • FIG. 7 shows the cumulative f a [%] to the right vertical axis (y 2 axes).
  • the analysis by the PIA-PTV was performed in a specified region (0.92 mm in width, 0.7 mm in length) as a visual field from a position below 4.5 mm from right below the nozzle hole.
  • the particle velocity is lower when the ultrasonic wave from the ultrasonic transducer is applied to the Laval nozzle (ULA) than when the ultrasonic wave is not applied (non-ULA).
  • UUA Laval nozzle
  • the particle size becomes smaller, and “air resistance acting on the particles> inertial force acting on the particles”, the flow velocity decreases.
  • the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
  • FIG. 10 is a conceptual diagram showing an example of the freezing apparatus 200 according to the embodiment of the present invention.
  • the freezing device 200 includes a support device 70 that supports the cryopreservation container 60.
  • the support device 70 includes a support portion 71, a spray position changing portion 72 (spray position changing means), and the like.
  • the support part 71 is detachable from the cryopreservation container 60 on the downstream side of the spray flow containing the cryogenic fine solid particles sprayed from the nozzle 1 (spiral nozzle 18) of the cryogenic fine solid particle continuous production apparatus as the spraying part.
  • the support portion 71 has a gripping portion 71a for gripping the cryopreservation container 60 at the tip portion.
  • the shape of the gripping portion 71a may be any shape as long as the cryopreservation container 60 can be detachably supported.
  • the gripping portion 71a has a substantially C-shaped cross section.
  • the cryopreservation container 60 may have an arbitrary shape such as a cylindrical shape.
  • the cryopreservation container 60 is formed in a round bottomed cylindrical shape so that it can be cooled with high efficiency without freezing unevenness, and a suspension in which cells 61 are suspended in a cryopreservation solution. And has a structure that can be sealed by a lid.
  • the spray position changing unit 72 changes the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support part 71 in order to reduce freezing unevenness for the cryopreservation container and shorten the freezing time.
  • a drive unit 81 such as a motor is connected to the spray position changing unit 72, and the cryopreservation container 60 supported by the support unit 71 is controlled by the control unit 82 in the vertical direction, the horizontal direction, It is configured to be movable in a predetermined direction such as the front-rear direction and the axial direction of the cylindrical cryopreservation container.
  • the spray position changing unit 72 is configured to be able to rotate the cryopreservation container 60 supported by the support unit 71 with a predetermined rotation direction, for example, the axial direction of the cylindrical cryopreservation container as the rotation axis.
  • the spray position changing part 72 may change the spray position to the cryopreservation container by changing the spray angle from the nozzle of the cryogenic fine solid particle continuous generation device.
  • the control unit 82 comprehensively controls each component of the freezing apparatus 200.
  • the control unit 82 is configured to be able to control the spray flow speed, pressure, spray time, and the like of the cryogenic fine solid particle continuous generation device of the freezing device 200.
  • the control part 82 controls the spray position to a cryopreservation container suitably by the spray position change part 72.
  • the freezing device includes a temperature detection unit such as a temperature sensor for detecting the temperature of the cryopreservation container and an infrared camera, and sprays the control unit 82 according to the temperature of the cryopreservation container detected by the temperature detection unit. You may be comprised so that the spraying time by the control of the spray position by the position change part 72, the cryogenic fine solid particle continuous production
  • A549 cells are human alveolar basal epithelial adenocarcinoma cells.
  • FIG. 11 is a diagram showing an example of a micrograph of a cell (A549 cell).
  • A549 cells are prepared (harvested) as cells to be frozen.
  • the concentration is 1 ⁇ 10 6 cells / mL with 5.08 ⁇ 10 6 cells and 4 ml of RPMI 1640 medium.
  • This cryopreservation container is a round-bottomed cylindrical container having a diameter of about 10 mm, a capacity of 1.8 mL, and an outer cap type.
  • the cryopreservation container may be formed of a resin material or metal material for cryopreservation having high thermal conductivity and high low-temperature strength.
  • a cryopreservation container containing a suspension obtained by directly suspending the above cells in a small amount of cryopreservation solution is supported by a support part of a freezing device, and a cryogenic fine solid particle is continuously produced by a cryogenic fine solid particle continuous production device.
  • a high-speed spray flow containing is continuously sprayed on a cryopreservation container containing cells and the like, and the cells in the cryopreservation container are rapidly vitrified and frozen.
  • CELLBANKER 1 plus from Nippon Zenyaku Kogyo Co., Ltd. is used as the cryopreservation solution, and the amount of the cryopreservation solution used is 1.5 mL per ampoule.
  • the components of the cryopreservation solution are defined as shown in Table 1.
  • a suspension in which cells are directly suspended in a cryopreservation solution at a pressure of about 0.252 to 0.253 MPa in the liquid nitrogen N 2 tank and a pressure of about 0.33 to 0.44 MPa in the vicinity of the injection nozzle A high-speed spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed to each stored cryopreservation container at a spraying time of 10, 20, 30, 45, 50, 60, 90, and 120 seconds.
  • the cells were vitrified and frozen rapidly, and then immersed in liquid nitrogen and stored.
  • a cryogenic fine solid nitrogen particle was stored in a cryopreservation container by being immersed in liquid nitrogen without spraying the cryopreservation container (spray time 0 second).
  • the cryotube (cryopreservation container) containing the vitrified cryopreserved cells is thawed in a water bath at a temperature of 37 ° C. Then, after pipetting 5 times, each is diluted with 9 mL of RPMI 1640 medium. Next, the centrifuge is centrifuged at 1500 rpm for 2 minutes. Then, the medium is removed by suction and suspended in 2 ml of RPMI 1640 medium. 10 ⁇ L of this suspension and 10 ⁇ L of trypan blue (BioRad) as a staining agent were mixed by pipetting, and the number of cells was counted with a cell counter. Table 2 shows an example of the experimental results of the spraying time and cell viability (%) of the cryogenic fine solid nitrogen particles.
  • FIG. 12 is a diagram showing an example of spraying time and cell survival rate (%) of cryogenic fine solid nitrogen particles.
  • the horizontal axis represents the spray time (sec) of the cryogenic fine solid nitrogen particles
  • the vertical axis represents the cell viability.
  • FIG. 12 and Table 2 the cell viability when the container containing the cells was cooled by simply immersing in liquid nitrogen without spraying a high-speed spray flow containing cryogenic fine solid nitrogen particles is shown in FIG. Shown as a value.
  • a spray flow containing cryogenic fine solid nitrogen particles is continuously sprayed for a predetermined number of seconds by the cryogenic fine solid particle continuous production apparatus 100 as a spraying section according to an embodiment of the present invention, and the cells in the container are vitrified.
  • the freezing method of the freezing apparatus increased the cell viability by about 23% (spray time 90 seconds).
  • the longer the spray time the higher the cell viability. It is considered that the longer the spray time, the higher the cooling rate, and the cells vitrify and freeze in high quality in a short time.
  • the freezing apparatus 200 freezes an elastic body membrane containing water such as the cells 61 as a freezing object.
  • This freezing apparatus includes a cryopreservation container 60 for storing a target to be frozen, and a cryopreservation object stored in the cryopreservation container 60 by continuously spraying the cryopreservation container 60 with a spray flow containing cryogenic fine solid particles at a high speed. And a cryogenic fine solid particle continuous production apparatus 100 as a spraying section for vitrification and freezing.
  • the cryogenic fine solid particle continuous generation apparatus 100 applies the nebulized stream containing the cryogenic fine solid particles to the cryopreservation container 60 containing the object to be frozen such as cells and the nebulized stream containing the cryogenic fine solid particles.
  • the object to be frozen such as the cells 61 housed in the cryopreservation container 60 is put into a glass frozen state by a synergistic effect of impact heat transfer, convection heat transfer, and latent heat of vaporization heat transfer.
  • the freezing device suppresses ice nucleation in the cell and rapidly freezes the cell in a glass state.
  • the maximum value of the freezing rate by continuous spraying of the high-speed spray flow containing the cryogenic fine solid nitrogen particles in the freezing apparatus according to the embodiment of the present invention is about ⁇ 25.8 K / Sec (a 25.8 ° C. drop per second). And can be rapidly cooled to about 63 K ( ⁇ 21.15 ° C.). Since new cryogenic fine solid particles always collide with the cryopreservation container, the freezing rate is maintained at a high speed, and the vitrification freezing of the cells is completed in a short time. Thereafter, the cryopreservation container containing the vitrified and frozen cells is immersed in liquid nitrogen and stored for a relatively long time.
  • the cells can be thawed with a high quality while suppressing renucleation by rapidly warming the cells with the thawing solution contained in the cryopreservation container 60. As described above, high cells Survival rate.
  • freezing is achieved by the synergistic effect of collision heat transfer, convection heat transfer, and latent heat of vaporization heat transfer. It is possible to provide a freezing device 200 that rapidly vitrifies and freezes objects such as cells in a storage container. Further, since the cells to be frozen are vitrified and frozen in a state of being stored in a cryopreservation container, no impurities are mixed during freezing and there is no damage due to a high-speed spray flow. As a comparative example, for example, there is a possibility that impurities are mixed in a method of cooling by directly immersing cells in liquid nitrogen.
  • cryopreservation liquid even when only a very small amount of the cryopreservation liquid is added to the cells to be frozen contained in the cryopreservation container 60, a high-speed spray flow containing cryogenic fine solid particles is continuously sprayed onto the cryopreservation container 60.
  • a high-speed spray flow containing cryogenic fine solid particles is continuously sprayed onto the cryopreservation container 60.
  • the particle number density of the cryogenic fine solid nitrogen particles (SN 2 particles) is increased, the particle speed is increased, etc. Improvement of the cooling capacity, specifically, the temperature drop rate can be increased, and the cells can be vitrified and frozen in a short time.
  • the freezing apparatus 200 is supported on the downstream side of the spray flow including the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous production apparatus 100 using the cryopreservation container 60 as a spraying unit.
  • a spray position changing portion 72 spray position changing means for changing the spray position of the spray flow with respect to the cryopreservation container 60 supported by the support portion 71.
  • the cryopreservation container 60 containing the cells to be frozen is supported by the support unit 71 on the downstream side of the spray flow containing the cryogenic fine solid particles ejected from the cryogenic fine solid particle continuous generation device 100, and the spray position
  • the changing portion 72 changes the spray position of the spray flow with respect to the cryopreservation container 60 so as to reduce the freezing unevenness with respect to the object to be frozen in the cryopreservation container 60.
  • the object to be frozen such as cells in the cryopreservation container is vitrified and frozen in a short time without unevenness of freezing. can do.
  • the spray position changing unit 72 is configured such that the cryopreservation container supported by the support unit 71 can be moved in a predetermined direction such as an up-down direction, a left-right direction, and a front-rear direction, and can be rotated in a predetermined rotation axis direction.
  • the spray position with respect to the cryopreservation container can be easily shifted.
  • the axis of the elongated cylindrical container may be rotated about the rotation axis.
  • the jet flow from the cryogenic fine solid particle continuous generation device 100 is inclined with respect to the side surface of the container.
  • the freezing speed increases and the freezing time can be shortened.
  • the jet flow from the cryogenic fine solid particle continuous production apparatus 100 is continuously sprayed on the side surface of the elongated cylindrical cryopreservation container obliquely at a high speed obliquely to the side surface of the container, and the elongated cylindrical shape is formed.
  • the freezing speed can be further increased and the freezing time can be further shortened.
  • cryogenic fine solid particles used in the freezing apparatus are configured by any one of nitrogen, carbon dioxide, argon, hydrogen, or a combination of two or more.
  • nitrogen as the cryogenic fine solid particles, cells and the like can be vitrified and frozen with low cooling cost and high efficiency.
  • the spray flow of cryogenic fine solid nitrogen particles has a very high cold enthalpy (cooling ability) among existing refrigerants, is harmless to living tissues, and is suitable as a refrigerant for freezing glass by high-speed rapid cooling .
  • the freezing object of the freezing apparatus according to the embodiment of the present invention can include iPS cells, ES cells, blood, plant cells, food (eg, fish eggs), and the like, and the application range of the freezing object is wide.
  • cryopreserving foods such as frozen food with the freezing apparatus according to the embodiment of the present invention, the cryopreservation solution can be frozen in a trace amount or not at all.
  • the spray part of the freezing apparatus which concerns on embodiment of this invention is a one-component cryogenic fine solid particle continuous production
  • generation apparatus mixes a cryogenic supercooled liquid with a high-speed flow of a cryogenic gas composed of the same elements as the supercooled liquid, thereby producing a one-component mixed phase flow.
  • a Laval nozzle unit 11 that is provided downstream of the mixing unit 10 and generates a spray flow including cryogenic fine solid particles from a one-component mixed phase flow generated in the mixing unit.
  • the Laval nozzle portion 11 is provided on the downstream side of the introduction portion 11a and the introduction portion 11a for introducing the one-component mixed phase flow generated by the mixing portion 10, and has a smaller opening cross-sectional area than the opening cross-sectional area of the introduction portion 11a.
  • a diameter portion 11b, and an injection portion 11c that is provided on the downstream side of the reduced diameter portion 11b, has an opening cross-sectional area that is larger than the opening cross-sectional area than the reduced diameter portion 11b, and has a shape that expands toward the downstream side.
  • the one-component mixed phase flow is adiabatically expanded in a state exceeding the speed of sound, and a spray flow including one-component cryogenic fine solid particles is continuously generated.
  • generation apparatus 100 can produce
  • the fine solid nitrogen spray generated by the one-component method (LN 2 -GN 2 ) is SN compared to the fine solid nitrogen spray generated by the two-component method (LN 2 -GHe) using cryogenic helium gas. Since the number density of two particles increases, the cooling effect is large.
  • the one-component (LN 2 -GN 2 ) cryogenic fine solid particle continuous generator is simple and inexpensive, and does not use cryogenic helium as a cryogen. Can be continuously generated.
  • the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 may include a spiral nozzle 18 at the tip of the Laval nozzle section 11.
  • the cryogenic fine solid particles can be further refined.
  • the cryogenic fine solid particle continuous generation apparatus 100 of the freezing apparatus 200 includes an ultrasonic transducer 6 that applies ultrasonic waves to the Laval nozzle unit 11.
  • an ultrasonic transducer 6 that applies ultrasonic waves to the Laval nozzle unit 11.
  • cavitation is generated in the one-component mixed phase flow in the Laval nozzle unit 11, promoting the ice nucleus generation of the cryogenic fine solid particles, and Further, it is possible to promote the miniaturization of one-component cryogenic fine solid particles having a substantially spherical shape with a fine uniform particle diameter.
  • the particle size of the cryogenic fine solid particles is reduced and the flow velocity is reduced.
  • the particle diameter is reduced, it is easy to evaporate, and it is difficult to form a film boiling state due to particle aggregation, and latent heat cooling using high-speed vapor phase change of solid particles is promoted. That is, under the ultrasonic wave application condition, the forced convection cooling effect is slightly reduced due to the decrease in the flow velocity, but the contact heat transfer and latent heat transfer characteristics are increased and the cooling effect is increased.
  • the cryogenic fine solid particle continuous production apparatus 100 of the freezing apparatus 200 has a heat insulating part 5 that vacuum-insulates all or part of the Laval nozzle part 11 with respect to the outside air. Specifically, the vicinity of the tip of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is accommodated in the vacuum heat insulating portion 5, and the temperature rise of the nozzle 1, the liquid nitrogen conduit 3, and the nitrogen gas conduit 4 is reduced. It has a structure. Therefore, a high-speed spray flow containing a cryogenic fine solid particle can be stably ejected from the Laval nozzle portion 11 of the nozzle 1 with a simple structure for a relatively long time.
  • the spray unit of the freezing device is a one-component cryogenic fine solid particle production continuous device, but is not limited to this form, and the spraying unit of the freezing device is a two-component cryogenic fine particle It may be a solid particle continuous production apparatus.
  • the two-component cryogenic fine solid particle continuous production apparatus may produce a high-speed spray flow containing cryogenic fine solid particles by cooling supercooled liquid nitrogen or the like with a refrigerant such as liquid helium, for example.
  • the cryopreservation container containing the object to be frozen is supported downstream of the spray flow by the support unit, and the cryogenic fine solid particle continuous generation apparatus 100 as the spray unit is frozen.
  • the spray container containing cryogenic fine solid particles continuously into the storage container 60 and vitrifying and freezing the object to be frozen stored in the cryopreservation container it is easy to achieve a high cell viability upon thawing. Furthermore, it is possible to rapidly vitrify an object to be frozen such as a cell.
  • the freezing apparatus continuously sprays the cryogenic container containing the cryogenic fine solid particles onto the cryopreservation container by the spraying unit to vitrify and freeze the object to be frozen contained in the cryopreservation container.
  • the freezing apparatus may spray the object to be frozen by vitrification by continuously spraying the object to be frozen directly with the spray flow containing the cryogenic fine solid particles by the spray unit.
  • the nitrogen (N 2) cryogenic fine solid particles continuously generating apparatus 100 using the may not in this form.
  • hydrogen (H 2 ), oxygen (O 2 ), argon (Ar), or the like may be employed.
  • a one-component mixed phase flow of cryogenic supercooled liquid hydrogen (GH 2 ) and cryogenic hydrogen gas (LH 2 ) is introduced into the Laval nozzle at a high speed, and the Laval nozzle is injected.
  • the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (the speed of sound of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
  • a one-component mixed phase flow of supercooled liquid oxygen (GO 2 ) and cryogenic oxygen gas (LO 2 ) at a low temperature is introduced into the Laval nozzle at a high speed, and injected into the injection part of the Laval nozzle.
  • the one-component multiphase flow is adiabatically expanded in a state exceeding the speed of sound (sound speed of the multiphase flow) to continuously generate a spray flow containing one-component cryogenic fine solid particles.
  • argon (Ar) When argon (Ar) is used, a one-component mixed phase flow of supercooled liquid argon and cryogenic argon gas at a low temperature is introduced into the Laval nozzle portion at high speed, and the one-component mixed phase flow is sonic ( Adiabatic expansion is performed in a state exceeding the sound velocity of the multiphase flow, and a spray flow containing one-component cryogenic fine solid particles is continuously generated.
  • the cryogenic temperature is a temperature lower than a general low temperature (0 ° C.) and a temperature equal to or lower than the boiling point at a standard pressure such as nitrogen, hydrogen, helium, or argon.
  • a standard pressure such as nitrogen, hydrogen, helium, or argon.
  • nitrogen (N 2 ) the temperature is about 77.36 K ( ⁇ 195.79 ° C.), which is the boiling point of nitrogen at standard pressure
  • hydrogen (H 2 ) is used.
  • the temperature is about 20.28 K ( ⁇ 252.87 ° C.) or less, which is the boiling point of hydrogen at the standard pressure
  • oxygen (O 2 ) oxygen
  • the boiling point of oxygen at the standard pressure is 90.2 K.
  • the temperature is about ( ⁇ 182.96 ° C.) or less, and when argon (Ar) is used, the temperature is about 83.80 K ( ⁇ 189.35 ° C.) or less.
  • a cryogenic supercooled liquid is used.
  • the present invention is not limited to this mode.
  • a cryogenic liquid that is not in a supercooled state may be used.
  • a cryogenic supercooled liquid it is possible to easily produce one-component cryogenic fine solid particles in a short time.
  • vitrification freezing control of the cell by a super-high heat flow rate can be performed by utilization of the high-speed spray flow containing a cryogenic fine solid particle.
  • various cells such as iPS cells and ES cells with high survival rate can be frozen at high quality and rapidly frozen. Vitrification and cryopreservation technology can be established.
  • Ultra-high-speed glass freezing method that does not require cryoprotection solution and suppresses ice nucleation as much as possible with conventional iPS cell freezing method due to ultra-high heat flux cooling effect of micro / nano level cryogenic fine solid particles And can provide this new technology to the biochemical field, medical engineering field, and various medical industries.
  • this technology has a high contribution not only in the medical, medical engineering, and life science fields but also in a wide range of different industrial fields.

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Abstract

L'invention concerne : un congélateur pour la vitrification rapide d'un sujet devant être congelé tel que des cellules, à l'aide d'un flux de pulvérisation contenant des microparticules solides à ultra-basse température dans la congélation de manière à obtenir un taux élevé de survie cellulaire à la décongélation ; et un procédé de congélation mettant en œuvre ce dernier. Le congélateur pour la congélation d'un sujet devant être congelé tel que des cellules comprend : un récipient de cryoconservation (60) qui loge le sujet devant être congelé tel que des cellules ; et une partie pulvérisation qui pulvérise en continu un flux de pulvérisation à grande vitesse contenant des microparticules solides à ultra-basse température sur le récipient de cryoconservation (60) de manière à congeler le sujet devant être congelé qui est logé dans le récipient de cryoconservation. Ce congélateur est pourvu d'une partie support (71) qui soutient le récipient de cryoconservation (60) et d'une partie de changement de position de pulvérisation (72) (moyen de changement de position du jet) qui change une position de pulvérisation du flux de pulvérisation à grande vitesse sur le récipient de cryoconservation soutenu par la partie support (71).
PCT/JP2016/058634 2015-03-27 2016-03-18 Congélateur et procédé de congélation WO2016158479A1 (fr)

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WO2021146122A1 (fr) * 2020-01-13 2021-07-22 The Regents Of The University Of California Dispositifs et procédés pour une surfusion à haute stabilité de milieux aqueux et de matière biologique

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CN112911941A (zh) * 2018-09-06 2021-06-04 可口可乐公司 使用高压气体的过冷饮料成核和冰晶形成

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WO2021146122A1 (fr) * 2020-01-13 2021-07-22 The Regents Of The University Of California Dispositifs et procédés pour une surfusion à haute stabilité de milieux aqueux et de matière biologique

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